This invention relates to quantitative determination of adenosine by immunoassay.
Adenosine has been well known in the art as one intermediate in the decomposition process of purine-type nucleic acid into nucleotide in living bodies and also as a substrate for the salvage reaction. Quite recently, based on some data, it has been suggested that adenosine may probably have some important physiological activities. Typical examples are as follows:
(1) It is probable that adenosine has a coronary circulation enhancing activity and physiologically controls the flow rate of coronary blood (Amer. J. Physiol. 204, p. 317, (1963)).
(2) Adenosine plays an important role in level controlling of cyclic AMP, and its effect is known to be contrariwise, namely positive or negative, depending on the types of tissues or cells (Proc. Natl. Acad. Sci. USA, 65, p. 1033 (1970); J. Biol. Chem., 247, p. 6866 (1972); Proc. Natl. Acad. Sci. USA, 74, p. 5482 (1977)).
Systems in which cyclic AMP is increased: Lymphocyte, brain slice, fibroblast, blood platelet, etc. PA1 Systems in which cyclic AMP is decreased: Lipocyte, renal cortex, hepatocyte, etc.
From these facts, it is considered probable that adenosine is secreted constantly from cells and functions to modify hormone actions.
(3) As to the possibility of association of congenital immunodeficiency with adenosine metabolism, the following hypothesis has been established from the discovery of defect of adenosinedeaminase in red blood cells of a hereditary disease patient who is substantially completely deficient in T and B cells. Deaminase defect.fwdarw.Adenosine increase.fwdarw.Pyrimidine metabolism abnormality.fwdarw.Lymphocyte growth inhibition.fwdarw.Immunodeficiency (Lancet, 2, p. 1067, (1972)).
As described above, adenosine is now considered not as a mere metabolism product but as an important physiologically active substance having a messenger function between cells which acts on some targets or receptors. Accordingly, measurement of the adenosine content in living bodies under various physiological conditions or disease conditions is expected to be of significance even in the field of clinical medicine in connection with prevention, diagnosis and therapy of diseases, to say nothing about the field of basic research of medicine.
In the prior art, as the method for measurement of adenosine, some methods different in principle have been proposed. As one practical method of quantitative determination of adenosine in a living body sample, the enzymatic method is known (Analytical Biochemistry, 95, p. 377 (1977)). According to this method, adenosine in a living body sample is deaminated with adenosinedeaminase to form inosine, which is then reacted with purine nucleoside phosphorylase in the presence of phosphoric acid to obtain hypoxanthine and subsequently hypoxanthine is reacted with xanthineoxidase, and the hydrogen peroxide formed is quantitatively determined by the fluorescent method employing peroxidase. However, this method requires a cumbersome quantitative determination procedure due to a large number of reagent additions as well as many reaction steps involved, and there is also an additional disadvantage such as low sensitivity with the minimum measurement limit of 20 pmol/tube.
As another quantitative determination method, the binding protein method has been reported (Analytical Biochemistry, 85, p. 132-138 (1978)). This is a kind of competitive protein binding assay, in which adenine analog-binding protein is prepared from rabbit red blood cells and allowed to react competitively with .sup.3 H-adenosine and the adenosine in a sample. This method also has a drawback in that pre-treatment is required for isolation of adenosine in a sample by PEI cellulose column chromatography in performing assay because of the low specificity of the binding protein.
On the other hand, in recent years, immunoassays such as radioimmunoassay, enzyme immunoassay and fluorescence immunoassay have been developed as methods for quantitative determination of a trace substance in living bodies and applied for determination of various substances in living bodies. The immunoassay is based on the principle to determine quantitatively a subject substance to be measured in a sample by allowing the subject substance and a predetermined amount of labelled ligands to react competitively with a predetermined amount of antibodies, and thereafter measuring the quantity of the label of the labelled ligands bound to the antibodies or free (unbound) labelled ligands. In the prior art, as the method for preparation of the antigen of adenosine and its antibody, it is known to treat adenosine with sodium periodate to cleave oxidatively the linkage between the 2'-position and the 3'-position in ribose residue, then, after neutralization of excessive periodic acid with ethylene glycol, couple the cleaved product with bovine serum albumin (BSA) at a pH of 9 to 9.5 and stabilize the binding between the hapten and the BSA by reduction with sodium borohydride to give an antigen, which is in turn inoculated into a rabbit for immunization purposes to produce an antibody (Proc. Natl. Acad. Sci. USA, 52, p. 68 (1964)). A radioimmunoassay making use of the antibody produced by the periodate method mentioned above is reported in Pfluegers Archiv., 370, p. 167 (1978). The radioimmunoassay needs a purification procedure for adenosine in biological samples such as chromatography because of the low specificity of the antibody. The present inventors attempted to apply the antibody thus produced to radioimmunoassay, and subjected adenosine to antigen-antibody reaction against the antibody. As a result, it was found that adenosine had substantially no binding ability with this antibody and therefore application of this antibody to quantitative determination of adenosine was impossible. Further, it was also found that the binding reaction with the antibody could occur to enable immunoassay, when adenosine in a sample to be measured or radiolabelled adenosine was subjected to the same treatment as in the preparation of the antigen, namely the periodic acid treatment and the ethylene glycol treatment. However, this method requires two steps for pre-treatment of a sample, and also gives rise to the problem of instability of adenosine in the radioactive ligand and sample, thus failing to serve as a practical quantitative determination method of adenosine.